The possibility of pre-birth microbiota colonisation remains controversial in the scientific community. Due to the placenta’s characteristics in pigs, the umbilical cord is the sole way for mother-foetus microbial transmission to occur. Studies on this topic have demonstrated conflicting results; some of these discrepancies might be due to differences during sampling, DNA extraction, bioinformatics and data analysis. The aim of this study is to assess a workflow for characterising the umbilical cord blood microbial profile by adjusting for the contaminating sources of bacterial DNA during the extraction procedure. The results show that among 735 amplicon sequence variants (ASVs), 568 ASVs were contaminants, while 165 ASVs were true samples. Using this workflow, we could distinguish the contaminant ASVs introduced during bacterial DNA extraction and amplification. With the results of the present study, however, we cannot confirm the pre-birth bacterial transfer by the umbilical cord blood due to the lack of samples representative of the contaminants in the surrounding sampling environment. Nevertheless, the present study can be used as a reference to address low microbial biomass, particularly with umbilical cord blood.